Walnut and pecan nut (also referred to as pecannut) are both edible nuts belonging to the family Juglandaceae and are edible foods. Since patients with the walnut allergen are increasing in Japan regarding walnut, the “walnut” is designated an item recommended to have an allergy label according to the Japan Standard Commodities Classification (the Ministry of Internal Affairs and Communications). Therefore, the development of a method for detecting the walnut allergen is urgently needed.
Meanwhile, pecan nut is not classified in walnut according to the Japan Standard Commodities Classification, although pecan nut is an edible nut belonging to the same family Juglandaceae.
The Japan Standard Commodities Classification
Walnut: caryopsis group→other caryopsis group→walnut
Pecan nut: caryopsis group→other caryopsis group→caryopsis group not classified in other groups.
Because the two are different in their classifications as described above, it is needed to discriminate (distinguish) the two from each other. Although the two are commonly allergic, it is recommended that only walnut should be labeled with such allergen indication. When an allergy occurs, it is now needed to identify the allergy whether the allergen is derived from walnut or pecan nut.
Although the walnut allergen ELISA detection system recently developed was applied, both the allergens of walnut and pecan nut were detected, so that the two could not be detected in a discriminative manner from each other. By the immunochemical method, as described above, it was very difficult to specifically detect walnut alone. As a method for detecting the walnut allergen in a food, the method is very advantageous for patients with the walnut allergy. From the viewpoint of food labels and following the recent increase of the import of pecan nut in Japan, however, the need of a method for detecting walnut and pecan nut in a discriminative manner from each other is further increasing.
An European research team reported a method for specifically detecting walnut by PCR (Polymerase Chain Reaction), recently (non-patent reference 1). However, the method is based on the real-time PCR of a capillary type but is not based on the block-type PCR as a standard method for use in food analyses in Japan. Food analyses by PCR in Japan are mainly done by PCR of the block type with a specific gene as the target. Using the method, methods for detecting wheat, buckwheat, peanut, soybean and kiwifruit (non-patent reference 2) have already been established.
The present inventors newly designed primers capable of specifically detecting edible vegetables with a possibility of the occurrence of food allergy, on the basis of the chloroplast matK (maturase-encoding gene) thereof and then successfully detected walnut in samples by PCR, using these primers (patent reference 1).
The method developed by the inventors is excellent because in addition to the walnut DNA of the genus Juglans of the family Juglandaceae of the order Fagales, demon walnut DNA and Persian walnut DNA of the same genus and Sawa-walnut DNA of the genus Protecaria of the same family can be detected by the method. However, it is strongly suggested that hickory DNA of the genus Carya of the same family of the same order, hazel nut DNA of the genus Corylus of the family Betulaceae of the same order, chestnut DNA of the genus Castanea of the family Fagaceae of the same order as close species, and DNAs of other biological species including other plants are never detected by the method.
Even in accordance with the invention of the previous application, further, no description is made about the discriminative detection of edible walnut from edible pecan nut of the genus Carya of the family Juglandaceae of the order Fagales as a close species. The present invention first achieved successfully the discriminative detection of the two in an accurate and simple manner.
The plants are classified as follows.    Edible walnut: Fagales (order); Juglandaceae (family); Juglans     Pecan: Fagales (order); Juglandaceae (family); Carya     Hazel: Fagales (order); Betulaceae (family); Corylus 
The information about the target gene (matK) is as follows. Herein, accession numbers in the GenBank are represented in the parentheses.    Walnut: Juglansx nigra (AF118036), Juglans californica (AF118027)    Pecan: Carya tomentosa (AF118039)    Hazel: Corylus ubelluna (AY373445)    Edible pecan: Carya illinoensis with no registered gene information    Non-patent reference 1: Eur. Food Res. Technol., 223, 373-377 (2006)    Non-patent reference 2: J. Food Chem., 30, 215-233 (2006)Patent reference 1: JP-A-2006-333729